The present invention relates to compositions and methods for detoxification or degradation of mycotoxins. The method has broad application in agricultural biotechnology, crop agriculture, and in the improvement of food grain quality.
Fungal diseases are common problems in crop agriculture. Many strides have been made against plant diseases as exemplified by the use of hybrid plants, pesticides, and improved agricultural practices. However, as any grower or home gardener can attest, the problems of fungal plant disease continue to cause difficulties in plant cultivation. Thus, there is a continuing need for new methods and materials for solving the problems caused by fungal diseases of plants.
These problems can be met through a variety of approaches. For example, the infectious organisms can be controlled through the use of agents that are selectively biocidal for the pathogens. Another method is interference with the mechanism by which the pathogen invades the host crop plant. Yet another method, in the case of pathogens that cause crop loss, is interference with the mechanism by which the pathogen causes injury to the host crop plant. In the case of pathogens that produce toxins that are undesirable to mammals or other animals that feed on the crop plants, interference with toxin production, storage, or activity can be beneficial.
Within the Gibberella species are several important pathogens which attack corn and other cereal crops in various countries. In corn, Gibberella is known to cause root, stem, and ear rot that result in severe crop reduction. The etiology of Gibberella ear mold is poorly understood, although physical damage to the ear and certain environmental conditions can contribute to its occurrence (Nelson et al. (1992) Mycopathologia 117:29-36). Gibberella maybe isolated from most field grown maize, even when no visible mold is present. Gibberella species that infect plants produce mycotoxins that may accumulate in infected plants or in stored grains, presenting serious health consequences for livestock and humans consuming grains and grain-derived products. The potential for transmission of these mycotoxins into eggs and meat has also been demonstrated (D""Mello et al. (1999) Anim. Feed Sci. and Tech. 80:183-205). Gibberella infection has been associated with chronic or acute mycotoxicoses in both farm animals and man.
Certain Gibberella species produce a mycotoxin, zearalenone, a non-steroidal estrogen. Zearalenone, produced mainly by Gibberella zeae (anamorph Fusarium graminearum), occurs in Gibberella-infected corn, wheat, barley, and other cereal crops. Zearalenone has been detected in hay, feed, corn, sorghum, dairy rations and barley that caused toxicosis in livestock in various countries (Ueno et al. (1985) CRC Critical Rev. Toxicol. 14:99). Zearalenone causes reproductive disorders in farm animals thereby compromising livestock health and productivity (Vanyi et al. (1995) Maygar Allatorvosok Lapja 50:424-430; Diekman and Green (1992) J. Anim. Sci. 50:1615-1627; Dacasto et al. (1995) Vet. Hum. Toxicol. 37:359-361; Etienne and Dourmad (1994) Livestock Production Science 40:99-113). For example, when consumed by swine, zearalenone may incite a hyperestrogenic response, including infertility, edematous swelling and reddening of the vuvla, reduced litter size, weak piglets, and congenital lesions of the external genitalia in piglets (Dacasto et al. (1995) Vet. Hum. Toxicol. 37:359-361; Chang et al. (1979) Am. J. Vet. Res.40:1260-1267). It is also physiologically active in cattle, rats, mice, guinea pigs, poultry, and plants (U.S. Pat. Nos. 5,962,304 and 6,074,838). Zearalenone has been shown to be teratogenic in rats (Ueno et al. (1976) Cancer Res. 36:445; Ueno et al. (1978) Cancer Res. 38:536), and to induce modulation of uterine tissues in mice (Ueno et al. (1970) Jap. J. Exp. Med. 45:199).
Among farm animals, pigs and sheep are most sensitive to dietary zearalenone. However, reproductive distortions due to consumption of zearalenone-contaminated feedstuff have been described in other animals including cattle (Coppock et al. (1990) Vet. Hum. Toxicol. 32:246-248) and mink (Bursian et al. (1992) J. Appl. Toxicol. 12:85-90). These citations are herein incorporated by reference.
Zearalenone has also been detected in commercially available cereal-based foods including baby foods and beer (Schollenberger et al. (1999) Mycopathologia 147:49-57 and Zollner et al. (2000) J Chromatogr B Biomed Sci Appl. 738:233-241). Zearalenone and/or its derivatives are genotoxic (Pfohl-Leszkowicz et al. (1995) Carcinogenesis 16:2315-2320), carcinogenic in mice (Grosse et al. (1997) Cancer Lett. 114:225-229; Battershill et al. (1998) Hum. Exp. Toxicol. 17:193-205), and induce hepatic carcinomas in the Armenian hamster (Coe, et al. (1992) Proc. Natl. Acad. Sci. 89:1085-1089). In humans, zearalenone exhibits estrogenic effects (Pitt (2000) Br. Med. Bull. 56:184-192), causes hyperestrogenic syndrome, particularly in children (Szuets et al. (1997) 5th European Fusarium Seminar, Proceedings:429-436), and at low concentrations, stimulates human breast cancer cells to enter the cell cycle (Dees et al. (1997) Environ Health Perspect 105:633-636). Each reference is herein incorporated by reference
Accordingly, there is a need in the art for novel methods with which zearalenone may be eliminated from a plant or harvested grain.
The present invention provides compositions and methods for the detoxification of zearalenone and structurally related mycotoxins. In particular, the present invention provides nucleotide sequences encoding zearalenone esterase polypeptides. More specifically, the present invention provides the nucleotide sequences ZES1 (SEQ ID NO:1), ZES2 (SEQ ID NO:3), ZES2b (SEQ ID NO:5) or biologically active variants thereof. Also provided are amino acid sequences (SEQ ID NOS:2,4, 6 and 7) and biologically active variants thereof encoded by the ZES1, ZES2 and ZES2b nucleotide sequences of the invention. The amino acid sequence set forth in SEQ ID NO:6 and 7 is the predicted amino acid sequence for ZES2b
Further compositions of the invention include expression cassettes and vectors for the expression of the sequences in plants and other organisms. Transformed plants, plant tissues, and seed, as well as, other transformed host cells are provided.
The invention further provides a method for detoxification of a mycotoxin in a plant. The method comprises stably incorporating into the genome of a plant a nucleotide sequence encoding a polypeptide of the invention operably linked to a promoter active in the plant. Another aspect of the invention provides a method for detoxification of a mycotoxin in or on a plant, grain or processed grain, by applying a microorganism having stably incorporated a heterologous nucleotide sequence of the invention. Additionally, the present invention provides a method for detoxification of mycotoxins in plants, grains or processed grains intended for use in feed or food. The method comprises applying to plants or grains a composition comprising a ZES1, ZES2, or ZES2b polypeptide or biologically active variant thereof.
The invention further provides a method for expression of a mycotoxin-degrading polypeptide in a non-human animal cell or population of non-human animal cells. The method comprises stably incorporating into the genome of a non-human animal cell a nucleotide sequence encoding a polypeptide of the invention operably linked to a promoter active in the non-human animal cell. The transformed cells are then cultured using appropriate methods. The expression of a polypeptide having the ability to degrade a mycotoxin such as zearalenone and the structural analogs thereof may occur in vitro or in vivo. The non-human animal cells include, but are not limited to, animal cells of any non-human origin, including but not limited to, mammals such as swine, sheep, goats, mink, cattle, horses, canines, and rodents. In an embodiment of the invention, the polypeptide is preferentially expressed in digestive tract cells including, but not limited to, salivary gland cells, liver cells, and gall bladder cells. The expressed polypeptide may be secreted from the non-human animal cell in which it is expressed. Secretion of the polypeptide may be improved or altered by operably linking a mammalian secretory signal to a nucleotide sequence of the invention.